HlyU is a transcription factor of the ArsR/SmtB family and activates the expression of the pathogenic Vibrio vulnificus RTX toxin. In contrast to the other metal-responding ArsR/SmtB proteins, HlyU does not sense metal ions. To provide its structural information, we elucidated the crystal structure of HlyU from V. vulnificus CMCP6 (HlyU_Vv). The monomeric HlyU_Vv architecture of five alpha-helices and two beta-strands, some of which constitute a typical DNA-binding winged helix-turn-helix (wHTH) motif, is very similar to that of other transcription regulators. Nonetheless, the homo-dimeric HlyU_Vv structure shows several different, three-dimensional features in the spatial position and the detailed dimeric interaction, which were not observed in the modeling study based on the same protein family and sequence similarity.